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• W2148517811 abstract "Neuroinflammatory disorders (including human immunodeficiency virus-1 encephalitis, HIVE) are associated with oxidative stress and inflammatory brain injury, and excessive alcohol use can exacerbate tissue damage. Using a murine model of HIVE, we investigated the effects of alcohol abuse on the clearance of virus-infected macrophages and neuroinflammation. Severe combined immunodeficient mice were reconstituted with human lymphocytes, and encephalitis was induced by intracranial injection of HIV-1-infected monocyte-derived macrophages (HIV-1+ MDM). Animals were fed an ethanol-containing diet beginning 2 weeks before lymphocyte engraftment and for the entire duration of the experiment. Lymphocyte engraftment was not altered by ethanol exposure. Alcohol-mediated immunosuppression in ethanol-fed mice was manifested by a significant decrease in CD8+/interferon-γ+ T lymphocytes, a fivefold increase in viremia, and diminished expression of immunoproteasomes in the spleen. Although both groups showed similar amounts of CD8+ T-lymphocyte infiltration in brain areas containing HIV-1+ MDMs, ethanol-fed mice featured double the amounts of HIV-1+ MDMs in the brain compared to controls. Ethanol-exposed mice demonstrated higher microglial reaction and enhanced oxidative stress. Alcohol exposure impaired immune responses (increased viremia, decreased immunoproteasome levels, and prevented efficient elimination of HIV-1+ MDMs) and enhanced neuroinflammation in HIVE mice. Thus, alcohol abuse could be a co-factor in progression of HIV-1 infection of the brain. Neuroinflammatory disorders (including human immunodeficiency virus-1 encephalitis, HIVE) are associated with oxidative stress and inflammatory brain injury, and excessive alcohol use can exacerbate tissue damage. Using a murine model of HIVE, we investigated the effects of alcohol abuse on the clearance of virus-infected macrophages and neuroinflammation. Severe combined immunodeficient mice were reconstituted with human lymphocytes, and encephalitis was induced by intracranial injection of HIV-1-infected monocyte-derived macrophages (HIV-1+ MDM). Animals were fed an ethanol-containing diet beginning 2 weeks before lymphocyte engraftment and for the entire duration of the experiment. Lymphocyte engraftment was not altered by ethanol exposure. Alcohol-mediated immunosuppression in ethanol-fed mice was manifested by a significant decrease in CD8+/interferon-γ+ T lymphocytes, a fivefold increase in viremia, and diminished expression of immunoproteasomes in the spleen. Although both groups showed similar amounts of CD8+ T-lymphocyte infiltration in brain areas containing HIV-1+ MDMs, ethanol-fed mice featured double the amounts of HIV-1+ MDMs in the brain compared to controls. Ethanol-exposed mice demonstrated higher microglial reaction and enhanced oxidative stress. Alcohol exposure impaired immune responses (increased viremia, decreased immunoproteasome levels, and prevented efficient elimination of HIV-1+ MDMs) and enhanced neuroinflammation in HIVE mice. Thus, alcohol abuse could be a co-factor in progression of HIV-1 infection of the brain. The independent effects of alcohol and human immunodeficiency virus (HIV)-1 in the central nervous system (CNS) are multifaceted and complex. Profound neurocognitive deficits are observed in alcoholics and patients with HIV-1-associated dementia.1Rosenbloom MJ O'Reilly A Sassoon SA Sullivan EV Pfefferbaum A Persistent cognitive deficits in community-treated alcoholic men and women volunteering for research: limited contribution from psychiatric comorbidity.J Stud Alcohol. 2005; 66: 254-265PubMed Google Scholar, 2McArthur JC Brew BJ Nath A Neurological complications of HIV infection.Lancet Neurol. 2005; 4: 543-555Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar HIV-1 encephalitis [(HIVE) neuropathological correlate of HIV-1-associated dementia] is characterized by macrophage infiltration, diffuse activation of resident brain macrophages (microglia), productive HIV-1 infection of macrophages, and neuronal damage.3Adamson P Etienne S Couraud P Calder V Greenwood J Lymphocyte migration through brain endothelial cell monolayers involves signaling through endothelial ICAM-1 via a rho-dependent pathway.Immunology. 1999; 162: 2964-2973Google Scholar, 4Persidsky Y Gendelman HE Mononuclear phagocyte immunity and the neuropathogenesis of HIV-1 infection.J Leukoc Biol. 2003; 74: 691-701Crossref PubMed Scopus (143) Google Scholar Neuropathological examination of brain tissue from chronic alcoholics suggests that alcohol abuse results in neuronal degeneration, ranging from minor dendritic structural change and synaptic changes to neuronal cell death in the CNS.5Harper C The neuropathology of alcohol-specific brain damage, or does alcohol damage the brain?.J Neuropathol Exp Neurol. 1998; 57: 101-110Crossref PubMed Scopus (327) Google Scholar Postmortem examination of brain tissues from individuals with HIVE and alcohol abusers suggests similar neuropathological changes in both conditions.6Sullivan EV Pfefferbaum A Neurocircuitry in alcoholism: a substrate of disruption and repair.Psychopharmacology (Berl). 2005; 180: 583-594Crossref PubMed Scopus (352) Google Scholar It has been suggested that the CNS suffers the additive effects of alcohol abuse and HIV-1 infection7Ashida N Arai H Yamasaki M Kita T Distinct signaling pathways for MCP-1-dependent integrin activation and chemotaxis.J Biol Chem. 2001; 276: 16555-16560Crossref PubMed Scopus (173) Google Scholar, 8Meyerhoff D Bloomer C Cardenas V Norman D Weiner M Fein G Elevated subcortical choline metabolites in cognitively and clinically symptomatic HIV+ patients.Neurology. 1999; 52: 995-1003Crossref PubMed Google Scholar; however, mechanistic studies assessing combined CNS injury are lacking. Interestingly, microglial activation (an important neuropathological correlate of HIV-1-associated dementia) was noted in animals chronically exposed to alcohol.9Riikonen J Jaatinen P Rintala J Porsti I Karjala K Hervonen A Intermittent ethanol exposure increases the number of cerebellar microglia.Alcohol Alcohol. 2002; 37: 421-426Crossref PubMed Scopus (46) Google Scholar In vitro data and results obtained in animal models suggest that alcohol abuse could increase HIV-1 replication through yet unknown pathways.10Wang X Douglas SD Metzger DS Guo CJ Li Y O'Brien CP Song L Davis-Vogal A Ho WZ Alcohol potentiates HIV-1 infection of human blood mononuclear phagocytes.Alcohol Clin Exp Res. 2002; 26: 1880-1886Crossref PubMed Scopus (44) Google Scholar, 11Bagby GJ Stoltz DA Zhang P Kolls JK Brown J Bohm Jr, RP Rockar R Purcell J Murphey-Corb M Nelson S The effect of chronic binge ethanol consumption on the primary stage of SIV infection in rhesus macaques.Alcohol Clin Exp Res. 2003; 27: 495-502Crossref PubMed Scopus (64) Google Scholar, 12Kumar R Perez-Casanova AE Tirado G Noel RJ Torres C Rodriguez I Martinez M Staprans S Kraiselburd E Yamamura Y Higley JD Kumar A Increased viral replication in simian immunodeficiency virus/simian-HIV-infected macaques with self-administering model of chronic alcohol consumption.J Acquir Immune Defic Syndr. 2005; 39: 386-390Crossref PubMed Scopus (53) Google Scholar Recent studies suggest that peripheral adaptive immune responses, including virus-specific CTL, control HIV-1 replication in the CNS.13Kim WK Corey S Chesney G Knight H Klumpp S Wuthrich C Letvin N Koralnik I Lackner A Veasey R Williams K Identification of T lymphocytes in simian immunodeficiency virus encephalitis: distribution of CD8+ T cells in association with central nervous system vessels and virus.J Neurovirol. 2004; 10: 315-325Crossref PubMed Scopus (36) Google Scholar, 14Potula R Poluektova L Knipe B Chrastil J Heilman D Dou H Takikawa O Munn DH Gendelman HE Persidsky Y Inhibition of indoleamine 2,3-dioxygenase (IDO) enhances elimination of virus-infected macrophages in an animal model of HIV-1 encephalitis.Blood. 2005; 106: 2382-2390Crossref PubMed Scopus (137) Google Scholar It is plausible to suggest that alcohol impairs immune responses, leading to increased viral replication, enhanced neuroinflammation, and neurodegeneration. Combined effects of alcohol abuse and HIV-1 on the CNS are difficult to investigate because of multiple confounding factors in human studies. Therefore, we tested whether alcohol abuse could affect clearance of virus-infected macrophages from the brain in a murine model of HIVE. To address our hypothesis, we used our previously developed mouse model of HIVE in hu-PBL-NOD/SCID HIVE mice. This model recapitulates the cellular immune responses against HIV-1-infected brain macrophages that occur in humans during progressive disease.15Poluektova LY Munn DH Persidsky Y Gendelman HE Generation of cytotoxic T cells against virus-infected human brain macrophages in a murine model of HIV-1 encephalitis.J Immunol. 2002; 168: 3941-3949PubMed Google Scholar Hu-PBL-NOD/SCID mice with focally induced HIVE and fed an ethanol-containing diet, demonstrated inefficient elimination of HIV-1-infected cells from the brain, augmented viremia, and increased microglial responses, suggesting that alcohol abuse can exacerbate HIV-1 CNS infection. Monocytes and peripheral blood lymphocytes (PBLs) were obtained by countercurrent centrifugal elutriation of leukopheresis packs from HIV-1, HIV-2, and hepatitis B-seronegative donors. Monocytes were cultivated in suspension culture using Teflon flasks in Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO) supplemented with 10% heat-inactivated pooled human serum, 1% glutamine, 50 μg/ml gentamicin, 10 μg/ml ciprofloxacin (Sigma), and 1000 U/ml highly purified recombinant human macrophage colony-stimulating factor (a generous gift from Genetics Institute, Cambridge, MA). After 7 days in culture, MDMs were infected with HIV-1ADA at a multiplicity of infection of 0.01.16Gendelman HE Orenstein JM Martin MA Ferrua C Mitra R Phipps T Wahl LA Lane HC Fauci AS Burke DS Meltzer MS Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes.J Exp Med. 1988; 167: 1428-1441Crossref PubMed Scopus (625) Google Scholar Four-week-old male NOD/C.B-17 SCID mice were purchased from Jackson Laboratory (Bar Harbor, ME). Animals were maintained in sterile microisolator cages under pathogen-free conditions in accordance with ethical guidelines for care of laboratory animals at the University of Nebraska Medical Center and National Institutes of Health. Animals were weight-matched and randomly assigned to an alcohol-containing diet or control group. The experimental groups were fed a Lieber-DeCarli liquid diet (Dyets Inc., Bethlehem, PA) containing 4% (v/v) ethanol (providing ∼22% ethanol-derived calories ad libitum, whereas the control groups were fed an isocaloric diet lacking ethanol.17Jerrells TR Smith W Eckardt MJ Murine model of ethanol-induced immunosuppression.Alcohol Clin Exp Res. 1990; 14: 546-550Crossref PubMed Scopus (111) Google Scholar The animals were monitored twice weekly for their body weight. Ethanol levels were detected in peripheral blood twice a week by assay kit (Diagnostic Chemicals Ltd., Charlottetown, PE, Canada). Two initial experiments (n = 60, Table 1) were performed to establish the chronic ethanol murine HIVE model (pair-feeding with Lieber-DeCarli diet) and to evaluate levels/intensity of neuroinflammatory responses in animals inoculated stereotactically with HIV-1-infected MDMs without human PBL reconstitution. The animals were fed with ethanol diet 2 weeks before MDM intracerebral inoculation and for the entire duration of the experiment. HIV-1ADA-infected MDMs (3 × 105 cells in 5 μl) were injected intracerebrally.18Persidsky Y Limoges J McComb R Bock P Baldwin T Tyor W Patil A Nottet HS Epstein L Gelbard H Flanagan E Reinhard J Pirruccello SJ Gendelman HE Human immunodeficiency virus encephalitis in SCID mice.Am J Pathol. 1996; 149: 1027-1053PubMed Google Scholar These experiments assured the absence of ethanol effects on MDM viability or level of HIV-1 infection in MDMs.Table 1Testing of Ethanol Effects in Mouse Model for HIVETreatment groupsWeeks of sacrificeWeek 1 (n)Week 2 (n)Control19*Total number of mice used in the study. The number of mice in the first two experiments (without PBL reconstitution) is shown in parentheses. (13)26*Total number of mice used in the study. The number of mice in the first two experiments (without PBL reconstitution) is shown in parentheses. (17)Ethanol19*Total number of mice used in the study. The number of mice in the first two experiments (without PBL reconstitution) is shown in parentheses. (13)24*Total number of mice used in the study. The number of mice in the first two experiments (without PBL reconstitution) is shown in parentheses. (17)* Total number of mice used in the study. The number of mice in the first two experiments (without PBL reconstitution) is shown in parentheses. Open table in a new tab The third experiment (n = 28) was designed to investigate the role of excessive alcohol use in HIV-1 CNS infection, including human lymphocyte reconstitution, immune responses, and neuropathological alterations. All animals were injected intraperitoneally with rat anti-mouse CD122 (0.25 mg/mouse) 3 days before PBL transplantation and twice with rabbit anti-mouse asialo-GM1 antibodies (0.2 mg/mouse) (Wako, Richmond, VA) 1 day before and 3 days after PBL injection (20 × 106cells/mouse) to deplete mouse natural killer cells, and in turn, facilitate the engraftment of human lymphocytes. Animals were fed a Lieber-DeCarli diet 2 weeks before lymphocyte engraftment and for the duration of the experiment. HIV-1ADA-infected MDMs (3 × 105 cells in 5 μl) were injected intracerebrally14Potula R Poluektova L Knipe B Chrastil J Heilman D Dou H Takikawa O Munn DH Gendelman HE Persidsky Y Inhibition of indoleamine 2,3-dioxygenase (IDO) enhances elimination of virus-infected macrophages in an animal model of HIV-1 encephalitis.Blood. 2005; 106: 2382-2390Crossref PubMed Scopus (137) Google Scholar 8 days after PBL reconstitution, thus generating hu-PBL-NOD/SCID HIVE mice. Mice were sacrificed at 7 and 14 days after intracerebral injection with human MDMs. PBLs were evaluated by flow cytometry for engraftment, and plasma was assayed for HIV-1 p24 antigen levels by enzyme-linked immunosorbent assay. A total of three independent experiments were performed using three different human leukocyte donors. Two-color FACS analysis was performed on blood and splenocytes at weeks 1 and 2 after intracerebral injection of human MDMs. Cells were incubated with fluorochrome-conjugated monoclonal antibodies (mAbs) specific to human CD4, CD8, CD56, CD3, and interferon (IFN)-γ (eBioscience, San Diego, CA) for 30 minutes at 4°C. To evaluate the cellular immune response, IFN-γ intracellular staining was performed in combination with anti-human CD8 and fluorescein isothiocyanate-conjugated anti-mouse CD45 antibodies (Abs) to exclude murine cells. Staining was performed as per the recommendation of the National Institutes of Health/National Institute of Allergy and Infections Disease, National Tetramer Core Facilities (Atlanta, GA). Data were analyzed with a FACSCalibur system using CellQuest software (Becton Dickinson Immunocytometry System, San Jose, CA). Western blot assays were performed on whole cell protein extracts of spleen and human brain homogenates (basal ganglia) using primary Abs or α-actin. Briefly, 15 to 20 μg of lysate protein was loaded onto 1.5-mm-thick 4 to 15% gradient polyacrylamide precast gels (Bio-Rad, Hercules, CA) and was electrophoresed. Proteins from the gels were transblotted onto nitrocellulose membranes (0.45-μm pore size) at 60 V for 1 hour at room temperature. The membranes were blocked with superblock (Bio-Rad) containing 2% nonfat dry milk for 40 minutes at room temperature. Blots were incubated for 1 hour at room temperature with respective primary antibody diluted 1/10 in superblock solution in 20 mmol/L phosphate-buffered saline, pH 7.4, containing 0.1% Tween-20 (PBST). Primary antibody-reacted blots were washed in three 5-minute washes of PBST. Immunoreactive bands were detected by luminol detection kit (Pierce, Rockford, IL) after exposure to Kodak X-ray film (Eastman-Kodak, Rochester, NY). The bands were quantified densitometrically on GelExpert as arbitrary volume integration units using the Molecular Dynamics ImageQuant software (Sunnyvale, CA). Results were expressed as ratios of intensities for target proteins to α-actin for normalization. The following Abs were used for Western blotting: monoclonal Abs to LMP2 or LMP7 (Affinity Research Products, Mamhead, UK), CD45 (Santa Cruz Biotechnology, Santa Cruz, CA), and nitrotyrosine (Upstate Cell Signaling Solutions, Lake Placid, NY). Histopathology and image analyses were performed as described previously.14Potula R Poluektova L Knipe B Chrastil J Heilman D Dou H Takikawa O Munn DH Gendelman HE Persidsky Y Inhibition of indoleamine 2,3-dioxygenase (IDO) enhances elimination of virus-infected macrophages in an animal model of HIV-1 encephalitis.Blood. 2005; 106: 2382-2390Crossref PubMed Scopus (137) Google Scholar Briefly, brain tissue was collected at days 7 and 14 after intracerebral injection of MDMs, fixed in 4% phosphate-buffered paraformaldehyde, and embedded in paraffin or frozen at −80°C for later use. In brief, coronal brain sections were cut to identify the injection site of HIV-1-infected MDMs. Serial 5-μm-thick sections (n = 30 to 100) covering the entire area of human MDM injection were cut for each mouse, and three to seven matched sections (10 sections apart) were analyzed. Immunohistochemical staining was performed using a basic indirect staining protocol. After deparaffinization, slides were heated for 30 minutes in 0.01mol/L citrate buffer at 95°C for antigen retrieval. Anti-vimentin mAb (1:50, clone 3B4; DAKO, Carpinteria, CA), which identifies all human leukocytes, was used to detect human cells in mouse brains. Human MDMs and CD8+ T lymphocytes were detected with CD68 (1:50 dilution, clone KP 1) and CD8 mAbs (1:50 dilution, clone 144B), respectively. Virus-infected cells were detected with mAb to HIV-1 p24 (1:10, clone Kal-1; DAKO). Reactive murine microglial cells were detected with ionized calcium-binding adapter molecule 1 (Iba1) antibody (1:500, Wako). Reactive astrocytes were labeled with antibodies to glial fibrillary acidic protein (GFAP) (1:1500, DAKO). Primary antibodies were detected with the appropriate biotinylated secondary antibodies and visualized with avidin-biotin complexes (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA) or horseradish peroxidase-coupled dextran polymer (EnVision, DAKO). Immunostained sections were counterstained with Mayer's hematoxylin. Deletion of primary antibody or irrelevant IgG isotype served as controls. The numbers of CD8+ lymphocytes, CD68+ MDMs, and HIV-1 p24+ MDMs in each section were counted in a blinded manner by three independent observers. Mean number of stained cells per section within the injected hemisphere was calculated for each mouse (three to seven sections/mouse) and a total of 13 to 17 mice per group were examined at weeks 1 and 2 after MDM injection. Light microscopic examination was performed with an Eclipse 800 microscope (Nikon Instruments Inc., Melville, NY) equipped with ×20/0.5 objective lens. Images were acquired with a digital CCD Color View II camera (Soft Imaging Systems, Lakewood, CO). Semiquantitative analysis (percentage of area occupied per section by immunostaining) for Iba1 and GFAP were performed by computer-assisted image analysis (Image-ProPlus; Media Cybernetics, Silver Spring, MD).14Potula R Poluektova L Knipe B Chrastil J Heilman D Dou H Takikawa O Munn DH Gendelman HE Persidsky Y Inhibition of indoleamine 2,3-dioxygenase (IDO) enhances elimination of virus-infected macrophages in an animal model of HIV-1 encephalitis.Blood. 2005; 106: 2382-2390Crossref PubMed Scopus (137) Google Scholar Data were analyzed using Prism (GraphPad) with Student's t-test for comparisons and analysis of variance. P values <0.05 were considered significant. All results are presented as mean plus or minus (±) SEM. The goal of this study was to investigate whether chronic alcohol abuse is a co-factor for progression of HIVE. To that end, hu-PBL-NOD/SCID HIVE mice were fed either Lieber-DeCarli liquid diet containing 4% ethanol or an isocaloric control diet. The mice fed an ethanol diet maintained a blood alcohol level of 0.13 to 0.22% (data not shown). Mice were engrafted with human PBLs to generate hu-PBL-NOD/SCID animals. Eight days after lymphocyte engraftment, encephalitis was induced by stereotactic injection of syngeneic human HIV-1ADA-infected MDMs (derived from the same donor as PBLs) into the basal ganglia and caudate generating hu-PBL-NOD/SCID HIVE mice. FACS analysis of blood and spleen (weeks 1 to 2) was performed in control and ethanol-fed hu-PBL-NOD/SCID HIVE mice to assess the level of human lymphocyte engraftment. Similar numbers of human CD3+, CD4+, and CD8+ T lymphocytes were found in the blood and spleens of control and ethanol-fed mice, suggesting equal levels of engraftment in both the groups. Table 2 shows results of two-color staining for human CD3+, CD4+, and CD8+ cells in peripheral blood samples of control and ethanol-fed hu-PBL-NOD/SCID HIVE mice. Our results show that ethanol-containing diet did not change the level of lymphocyte engraftment in our model.Table 2Human Lymphocytes and Plasma Levels of HIV-1 p24 in the Peripheral Blood of Ethanol-Fed Versus Pair-Fed Control MiceWeeks*Days after MDM injection.Treatment groupsLymphocytes population (%) (mean ± SEM)HIV-1 p24 pg/ml (mean ± SEM)CD3+†No significant difference in percentage of CD3+/CD8+/CD4+ cells between the groups.CD4+†No significant difference in percentage of CD3+/CD8+/CD4+ cells between the groups.CD8+†No significant difference in percentage of CD3+/CD8+/CD4+ cells between the groups.Week 1Control44.99 ± 1.0615.32 ± 1.0822.25 ± 0.792.07 ± 0.59Ethanol44.21 ± 1.1316.27 ± 0.6718.02 ± 0.325.84 ± 1.08Week 2Control62.86 ± 0.3532.31 ± 0.3726.97 ± 0.2524.6 ± 14.43Ethanol65.01 ± 0.6830.53 ± 0.4631.32 ± 0.26132.13 ± 58.47‡P < 0.032, compared with control.* Days after MDM injection.† No significant difference in percentage of CD3+/CD8+/CD4+ cells between the groups.‡ P < 0.032, compared with control. Open table in a new tab Chronic ethanol abuse in humans leads to a variety of immunomodulatory events that can alter resistance to infectious agents. To study ethanol effect on viremia, HIV-1 p24 blood levels were measured in control and ethanol-fed hu-PBL-NOD/SCID HIVE mice. HIV-1 p24 levels in ethanol-fed mice were fivefold greater compared to control mice (132 ± 58.47 pg/ml in ethanol versus 24.6 ± 14.43 pg/ml in control mice, P < 0.03; Table 2). Immunosuppressive effects of alcohol abuse have been associated with ethanol-induced impairment of cellular responses. To determine whether alcohol exposure affects immune responses in our model, we compared levels of IFN-γ-positive effector CD8+ T lymphocytes in peripheral blood by FACS analysis (Figure 1A). The percentage of IFN-γ-positive cells was significantly lower in ethanol-fed mice (P < 0.05) compared to controls (Figure 1B). These results suggest that chronic ethanol administration diminished induction of anti-viral cellular immune responses. Proteasomes (PRs) are multicatalytic protease complexes that degrade polyubiquitinated cellular proteins. PR constitutive subunits have chymotrypsin-like (X), trypsin-like (Y), and peptidyl-glutamyl-peptide hydrolase (Z) activities.19Piccinini M Tazartes O Mostert M Musso A DeMarchi M Rinaudo MT Structural and functional characterization of 20S and 26S proteasomes from bovine brain.Brain Res Mol Brain Res. 2000; 76: 103-114Crossref PubMed Scopus (23) Google Scholar Stimulation of PRs constitutes subunits (X, Y, and Z) by proinflammatory cytokines such as IFN-γ, tumor necrosis factor-α, and interleukin-6 are, respectively, replaced by subunits LMP2, LMP7, and MELC-1 which are now referred as inducible PR subunits.20Fruh K Gruhler A Krishna RM Schoenhals GJ A comparison of viral immune escape strategies targeting the MHC class I assembly pathway.Immunol Rev. 1999; 168: 157-166Crossref PubMed Scopus (95) Google Scholar, 21Khan S van den Broek M Schwarz K de Giuli R Diener PA Groettrup M Immunoproteasomes largely replace constitutive proteasomes during an antiviral and antibacterial immune response in the liver.J Immunol. 2001; 167: 6859-6868PubMed Google Scholar PRs with inducible PR subunits are known as immunoproteasomes (IPRs), which process antigenic peptides for antigen presentation on major histocompatibility complex (MHC) class I molecules. We demonstrated that both HIV-1 and ethanol diminish PR activity and IPR content in MDMs, and therefore, may contribute to dysfunction in the HIV-infected host.22Haorah J Heilman D Diekmann C Osna N Donohue Jr, TM Ghorpade A Persidsky Y Alcohol and HIV decrease proteasome and immunoproteasome function in macrophages: implications for impaired immune function during disease.Cell Immunol. 2004; 229: 139-148Crossref PubMed Scopus (56) Google Scholar To further study the effects of ethanol on IPR content in vivo, we preformed Western blot analysis of IPR subunits, LMP2, and LMP7 on protein extracts derived from spleen. The IPR expression (LMP2 and LMP7) was adjusted to human CD45 (reflecting number of human cells) in the samples and expressed as the ratio of target protein immunoreactivity to that of internal standard α-actin. The levels of LMP2 and LMP7 were significantly lower as compared to mice on control diet (P < 0.04; Figure 1, C and D). These results indicate that chronic ethanol consumption affects IPR protein levels in vivo. Initial experiments were performed to establish the ethanol model (pair-feeding with Lieber-DeCarli diet) to evaluate the in vivo effects of ethanol on MDM viability and HIV-1 infection in nonreconstituted animals inoculated intracerebrally with HIV-1-infected MDMs (Table 1). Serial brain sections were immunostained for CD68 and HIV-1 p24. Mean numbers of stained cells per section within the injected hemisphere were calculated for each mouse (three to seven sections/mouse) and mean numbers of cells for 13 to 17 mice per each group were evaluated at weeks 1 and 2 after MDM injection. Equal numbers of MDMs were detected in the brain tissue of controls and ethanol-fed mice (72.2 ± 14.2 versus 64.6 ± 26.7 at week 1 and 46 ± 14.9 versus 32.6 ± 5 at week 2; P > 0.05) indicating that ethanol feeding did not significantly affect MDM viability in the brain (Figure 2A). Ethanol exposure did not significantly change the levels of HIV-1 infection in both groups (percentage of HIV-1 p24+ MDMs, 28 ± 4.5% versus 20.8 ± 2.4% at week 1 and 27.8 ± 5.1% versus 22.8 ± 13% at week 2, P > 0.05; Figure 2B). These experiments assured that ethanol feeding had no effect on HIV-1 MDM infection or cell viability in the brain. To analyze neuroinflammatory responses in the hu-PBL-NOD/SCID mice brain, serial sections were immunostained for human CD68 (marker for human MDMs), HIV-1 p24 (viral antigen), human CD8 (T cells), and Iba1 (mouse microglial marker). Cells were counted in a blinded manner by three investigators, and the number of positive cells was averaged for the area covering the entire left hemisphere (inoculated with MDMs). At week 1, comparable numbers of CD68+ MDMs (Figure 3, A and E) and CD8+ lymphocytes (Figure 3, C and G) were seen in the brains of control and ethanol mice. Ethanol-fed mice demonstrated more HIV-1 p24+ MDMs (Figure 3, B and F) and greater levels of microglial reaction (number of cells and activated phenotype; Figure 3, D and H) compared with controls. By week 2, brains of ethanol-fed mice contained more CD68+ MDMs (Figure 3, I and M) and HIV-1 p24+ MDMs (Figure 3, J and N). Enhanced microglial reaction (Figure 3, L and P) were seen in ethanol-fed animals compared with controls. Similar to week 1, numbers of CD8+ lymphocytes detected in brain tissues from both groups were comparable (Figure 3, K and O). To explore the effects of ethanol on elimination of virus-infected macrophages, CD68+, HIV-1 p24+, and CD8+ lymphocytes were counted in brain tissue in a blinded manner by three independent observers. Mean numbers of stained cells per section within the injection hemisphere were calculated for each mouse (three to seven sections/mouse) and the mean numbers of cells for six to nine mice per each group were evaluated at weeks 1 and 2 after MDM injection. As shown in Figure 4A, the mean numbers of CD68+ MDMs was lower, although not statistically significant from the control group, in the ethanol group (154 ± 34 versus 102.83 ± 20, P > 0.05) at week 1. At week 2 the amount of CD68+ MDMs in ethanol group was significantly higher than in the control mice (19 ± 7 versus 5 ± 3, P < 0.03). Although comparable numbers of CD8+ T cells were observed at weeks 1 and 2 in ethanol and control mice (Figure 4B), more HIV-1 p24+ MDMs were found in the ethanol mice at week 1 (42 ± 8 versus 21 ± 6; P < 0.04) and week 2 (7.57 ± 2.16 versus 3 ± 0.957, P < 0.02) as compared with control mice (Figure 4C). These results suggest inefficient clearance of HIV-1-infected p24+ MDMs (main target for CTL) from the brains of ethanol mice. Induction of an inflammatory response in the brain was determined by measurement of microglial reaction in brain tissue (assessed by digital image analysis and expressed as percentage of the area occupied by Iba1 immunostaining). Microglial reaction was threefold higher in nonreconstituted ethanol-fed animals (P < 0.008; Figure 5A) compared to cont" @default.
• W2148517811 created "2016-06-24" @default.
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• W2148517811 date "2006-04-01" @default.
• W2148517811 modified "2023-10-12" @default.
• W2148517811 title "Alcohol Abuse Enhances Neuroinflammation and Impairs Immune Responses in an Animal Model of Human Immunodeficiency Virus-1 Encephalitis" @default.
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